Sarcomere lengths are one of the main determinants of how a muscle contributes to behavior. Whether a muscle functions primarily for movement stabilization or for power generation is strongly influenced by the sarcomere lengths over which that muscle operates. Moreover, alterations in sarcomere properties contribute to muscle damage following injury. Although investigators have used a variety of methods to measure sarcomere lengths, the ability to measure these lengths in situ and how they vary when the limb is moved or when the muscle is active, remains a fundamental challenge. The research in this proposal will develop a promising new technique, two photon microscopy, to measure sarcomere lengths in muscles in situ. This technique will allow us to characterize sarcomere length variations across muscles, limb configurations, and activation patterns. Importantly, it also will allow us to begin characterizing how these measures change following exercise related muscle damage. This technique has several potential advantages over current techniques of characterizing sarcomere lengths of muscles. First, it can measure sarcomere lengths without disturbing muscle fibers, so sarcomere lengths can be measured in an intact muscle. Second, sarcomere lengths can be measured in functioning muscles, so that effects of activation can be directly evaluated. Such measurements are impossible with current techniques of measuring sarcomere lengths. Third, multiple sarcomere length measurements can be made quickly and repeatedly, so that we can examine sarcomere operating ranges directly without resorting to extrapolation. This repeatability also allows us to visualize the development of muscle damage during exercise related activity in situ; e.g. tracking sarcomere properties in the same muscle as damage is progressively induced through eccentric contractions. Finally, we will perform these measurements in an in vivo preparation that we have recently developed for measuring muscle actions, so that we will be able to measure both sarcomere lengths and muscle actions in situ. The ability to collect these measurements together in the same preparation will give us unprecedented insight into the function of a muscle and how it contributes to behavior. We have three specific aims in this proposal. First, we will measure the operating range of sarcomeres across a large number of muscles in the rat hindlimb, to determine the relationship between sarcomere lengths and hypothesized muscle functions. Second, we will measure the change in sarcomere operating ranges after muscle activation, to determine the degree to which neural control alters sarcomere operating ranges. Finally, we will image sarcomeres during the development of muscle damage induced by eccentric contractions. If successful, this research has the potential to directly address these fundamental issues of muscle function and disease, demonstrating the potential power of two photon microscopy in studies of muscle function.